Conventional techniques of molecular imprinting have provided useful methods for the preparation of matrices that are capable of selectively capturing a target molecule. To prepare a molecular imprint, a matrix is formed around a template molecule. After the matrix has formed and the template molecule has been removed, the resulting molecular imprint can then be used to selectively capture the template molecule. As early as 1949, a silica gel was created that selectively bound a dye (Dickey, 1949, Proc. Natl. Acad. Sci. USA 35:227–229). Recently, an imprint prepared with phenyl-α-D-mannopyranoside was sufficiently selective to resolve a racemic mixture of the saccharide (Wulff, 1998, Chemtech 28:19–26).
Current methods form imprints of template molecules in organic polymers (Wulff, 1998, supra). To create cavities of defined shape, polymerizable molecules are bound, covalently or noncovalently, to a template molecule (Wulff, 1998, supra). The resulting complex is then copolymerized in the presence of a large amount of a cross-linking reagent (Wulff, 1998, supra). The templates are then removed, leaving cavities having defined shapes (Wulff, 1998, supra). Molecular imprints made by such a technique display selective binding for the template molecule. Molecular imprints have been used for chromatographic separation, immunoassays, chemosensors, and even catalysis (Wulff, 1998, supra).
However, failings of conventional techniques limit the broad application of molecular imprints. According to a recent review, two issues “of great importance” that limit the application of conventional molecular imprints are their limited capacity and the heterogeneity of imprint cavities (Cormack and Mosbach, 1999, Reactive and Functional Polymers 41:115–124). When conventional imprints are used to capture the template molecule, it is believed that their random distribution of imprint cavities limits their accessibility to the template molecules. Typically, the majority of cavities are localized in the interior of the molecular imprint. These interior cavities are less accessible to the template molecule than cavities localized at the surface of the imprint. This effect not only reduces the number of cavities available for binding, but also limits the types of molecules that can be bound or captured. In particular, large molecules that cannot penetrate the matrix material of a molecular imprint can bind only at surface cavities. Thus, conventional molecular imprints are not advantageous for specifically capturing large molecules such as proteins, nucleic acids and other macromolecules.
The binding capacity of conventional imprints is also reduced by the random orientations of their cavities. In forming a molecular imprint by conventional techniques, the template molecules are randomly oriented within the matrix. Thus, the corresponding molecular imprint cavities are also randomly oriented. If a particular orientation of an imprint cavity binds a target molecule more efficiently than other orientations, then only the fraction of cavities that are properly oriented will display efficient binding. The random orientation of the cavities, combined with their random distribution throughout the imprint, exacerbates the poor binding capacity of conventional molecular imprints.
Finally, conventional techniques suffer from leakage of the template molecule (Wulff, 1999, supra). When the imprint is formed, many template molecules are trapped deep within the imprint matrix. Trapped template molecules that are not removed may leak during the use of the molecular imprint. Leakage of the template molecule contaminates the sample and hinders application of conventional molecular imprints, particularly applications that involve binding or capturing minute amounts of the template molecule. For instance, this shortcoming of conventional molecular imprints has particularly limited their application in the pharmaceutical industry (Wulff, 1999, supra).
What is needed are novel molecular imprints that overcome the shortcomings of conventional molecular imprints. Novel methods of making molecular imprints with oriented and accessible binding cavities, and less leakage of the template molecule, will have improved capacity, specificity, and application, particularly for large molecules such as proteins, nucleic acids and polysaccharides.